Parkinsons disease is a progressive condition such that patients initially show subtle cognitive deficits before gradually developing the more visible motor manifestations. We are pursuing two strategies to develop new insight into the causes and potential treatments of this disease. In the first, we pursue a deeper understanding of the developmental origin of dopamine neurons. In the second, we focus on the mechanisms that maintain the survival of dopamine neurons. 1. The first mouse model where dopamine neurons die spontaneously in old age identifies the Foxa2 gene as a central regulator of both the embryonic specification and the adult survival of dopamine neurons. By pursuing the developmental origin of dopamine neurons, we showed that the transcription factor foxA2 is responsible for their birth in early development their death in old age. The midbrain dopamine system is composed of different subsets of neurons that project to different regions of the forebrain and show different vulnerability to the etiological causes of Parkinsons disease. In the developing brain, the dopamine neurons originate from the midbrain floor plate. The floor-plate is characterized by the expression of sonic hedgehog, a morphogen that regulates the differentiation of adjacent, more lateral neurons. In a collaboration with Dr. R. Awatramani (Northwestern University, Chicago) we have used sonic hedgehog expression to target changes in gene expression to the developing midbrain. These experiments extend our initial identification of the floor plate as a source of dopamine neurons by further specifying three groups or phases of neuronal specification that generate (1) the rostral linear nucleus and the ventral tegmental area/interfascicular regions (VTA/IF) (2) the substantia nigra pars compacta (SNpc) and (3) the red nucleus, Edinger Westphal nucleus and the oculomotor nucleus. These results will help us understand the selective vulnerability of different types of dopamine neuron and provide a more precise specification of the neuron types that must be generated by the stem cell technologies we are developing. 2. In this project, we are currently focused on understanding the mechanisms that regulate the dopamine neurons. An understanding of spontaneous loss is the most valuable benefit of Foxa2 +/- mice we generated. But this approach is difficult because neuron loss does not occur until the mice are old. Chemical toxins that specifically kill dopamine neurons provide the most widely used model of Parkinsons disease. In the last year we reported that single injections of angiogenic growth factors have pronounced benefit in chemically lesioned animals. Remarkably, a single intra-ventricular injection of Notch ligands alone or in combination with other angiogenic factors promotes widespread activation of the stem cell niche in the adult brain and rescues dopaminergic neurons. These data suggest vascular cytokines promote regenerative responses to brain injury. As we discuss in NS002881-17, a major goal of our group is to establish new in vitro and in vivo assays to predict the activation of the stem cell compartment in primates. In this project, we pursue a deeper understanding of survival signaling in the stem cell niche in animal models. In the past, we established that chemically lesioned dopamine neurons show an immediate change that predicts the later loss of the cells. We have now established the chemical tests for the low molecular weight neurotransmitters that dominate signaling in the striatum, the target region for dopaminergic projections from the substantia nigra;including dopamine, serotonin, GABA, glutamate and acteyl choline. We now propose to measure the alteration in neurotransmitter levels caused by increasing doses of cytotoxins specific for dopamine neurons. The status of the Foxa2 survival signaling pathway will be used to determine status of dopamine neurons and the expression of the Hes3 gene will define the activation of stem cells that are known to physically interact with the axons and dendrites of dopamine neurons. By using this approach, we propose to define the changes in neuronal activity that precede and perhaps regulate the loss of dopamine neurons. We suggest that targeting the mechanism of neuron loss may be a more rapid and more secure route to a clinical treatment than cell replacement therapy.